L. Lacey Knowles

Delimiting Species without Monophyletic Gene Trees

Genetic data are frequently used to delimit species, where species status is determined on the basis of an exclusivity criterium, such as reciprocal monophyly. Not only are there numerous empirical examples of incongruence between the boundaries inferred from such data compared to other sources like morphology -- especially with recently derived species, but population genetic theory also clearly shows that an inevitable bias in species status results because genetic thresholds do not explicitly take into account how the timing of speciation influences patterns of genetic differentiation. This study represents a fundamental shift in how genetic data might be used to delimit species. Rather than equating gene trees with a species tree or basing species status on some genetic threshold, the relationship between the gene trees and the species history is modeled probabilistically. Here we show that the same theory that is used to calculate the probability of reciprocal monophyly can also be used to delimit species despite widespread incomplete lineage sorting. The results from a preliminary simulation study suggest that very recently derived species can be accurately identified long before the requisite time for reciprocal monophyly to be achieved following speciation. The study also indicates the importance of sampling, both with regards to loci and individuals. Withstanding a thorough investigation into the conditions under which the coalescent-based approach will be effective, namely how the timing of divergence relative to the effective population size of species affects accurate species delimitation, the results are nevertheless consistent with other recent studies (aimed at inferring species relationships), showing that despite the lack of monophyletic gene trees, a signal of species divergence persists and can be extracted. Using an explicit model-based approach also avoids two primary problems with species delimitation that result when genetic thresholds are applied with genetic data -- the inherent biases in species detection arising from when and how speciation occurred, and failure to take into account the high stochastic variance of genetic processes. Both the utility and sensitivities of the coalescent-based approach outlined here are discussed; most notably, a model-based approach is essential for determining whether incompletely sorted gene lineages are (or are not) consistent with separate species lineages, and such inferences require accurate model parameterization (i.e., a range of realistic effective population sizes relative to potential times of divergence for the purported species). It is the goal (and motivation of this study) that genetic data might be used effectively as a source of complementation to other sources of data for diagnosing species, as opposed to the exclusion of other evidence for species delimitation, which will require an explicit consideration of the effects of the temporal dynamic of lineage splitting on genetic data.

STEM: species tree estimation using maximum likelihood for gene trees under coalescence

UNLABELLED STEM is a software package written in the C language to obtain maximum likelihood (ML) estimates for phylogenetic species trees given a sample of gene trees under the coalescent model. It includes options to compute the ML species tree, search the space of all species trees for the k trees of highest likelihood and compute ML branch lengths for a user-input species tree. AVAILABILITY The STEM package, including source code, is freely available at http://www.stat.osu.edu/~lkubatko/software/STEM/. SUPPLEMENTARY INFORMATION Supplementary data are available at Bioinformatics online.

Did the Pleistocene glaciations promote divergence? Tests of explicit refugial models in montane grasshopprers

There is a long‐standing debate over whether or not the Pleistocene glaciations promoted speciation. While some models predict that extensive mixing of populations during interglacial expansion would have inhibited divergence, others postulate that divergence among allopatric glacial refuges or founder events during recolonization of previously glaciated areas would have promoted differentiation. Using a combination of traditional and coalescent based population genetic approaches, this study finds that the glaciations did not inhibit divergence among populations of the grasshopper Melanoplus oregonensis. Instead, drift associated with recolonization of previously glaciated areas, as well as divergence among multiple allopatric glacial refugia, have both contributed to differentiation in this montane grasshopper from the ‘sky islands’ of the northern Rocky Mountains. Significant population structure was detected by phylogenetic and FST analyses, including significant FST values among individual pairs of sky‐island populations. In addition to clustering of haplotypes within populations, there is some evidence of regional phylogeographic structure, although none of the ‘regional groups’ form a monophyletic clade and there is a lack of concordance between the genealogical and geographical positions of some haplotypes. However, coalescent simulations confirm there is significant regional phylogeographic structure that most likely reflects divergence among multiple ancestral refugial populations, and indicate that it is very unlikely that the observed gene tree could have been produced by the fragmentation of a single widespread ancestral population. Thus, rather than inhibiting differentiation, the glaciations appear to have promoted population divergence in M. oregonensis, suggesting that they may have contributed to the radiation of Melanoplus species during the Pleistocene.

Estimating Species Phylogeny from Gene-Tree Probabilities Despite Incomplete Lineage Sorting: An Example from Melanoplus Grasshoppers

Estimating phylogenetic relationships among closely related species can be extremely difficult when there is incongruence among gene trees and between the gene trees and the species tree. Here we show that incorporating a model of the stochastic loss of gene lineages by genetic drift into the phylogenetic estimation procedure can provide a robust estimate of species relationships, despite widespread incomplete sorting of ancestral polymorphism. This approach is applied to a group of montane Melanoplus grasshoppers for which genealogical discordance among loci and incomplete lineage sorting obscures any obvious phylogenetic relationships among species. Unlike traditional treatments where gene trees estimated using standard phylogenetic methods are implicitly equated with the species tree, with the coalescent-based approach the species tree is modeled probabilistically from the estimated gene trees. The estimated species phylogeny (the ESP) is calculated for the grasshoppers from multiple gene trees reconstructed for nuclear loci and a mitochondrial gene. This empirical application is coupled with a simulation study to explore the performance of the coalescent-based approach. Specifically, we test the accuracy of the ESP given the data based on analyses of simulated data matching the multilocus data collected in Melanoplus (i.e., data were simulated for each locus with the same number of base pairs and locus-specific mutational models). The results of the study show that ESPs can be computed using the coalescent-based approach long before reciprocal monophyly has been achieved, and that these statistical estimates are accurate. This contrasts with analyses of the empirical data collected in Melanoplus and simulated data based on concatenation of multiple loci, for which the incomplete lineage sorting of recently diverged species posed significant problems. The strengths and potential challenges associated with incorporating an explicit model of gene-lineage coalescence into the phylogenetic procedure to obtain an ESP, as illustrated by application to Melanoplus, versus concatenation and consensus approaches are discussed. This study represents a fundamental shift in how species relationships are estimated - the relationship between the gene trees and the species phylogeny is modeled probabilistically rather than equating gene trees with a species tree.

The burgeoning field of statistical phylogeography

In the newly emerging field of statistical phylogeography, consideration of the stochastic nature of genetic processes and explicit reference to theoretical expectations under various models has dramatically transformed how historical processes are studied. Rather than being restricted to ad hoc explanations for observed patterns of genetic variation, assessments about the underlying evolutionary processes are now based on statistical tests of various hypotheses, as well as estimates of the parameters specified by the models. A wide range of demographical and biogeographical processes can be accommodated by these new analytical approaches, providing biologically more realistic models. Because of these advances, statistical phylogeography can provide unprecedented insights about a species’ history, including decisive information about the factors that shape patterns of genetic variation, species distributions, and speciation. However, to improve our understanding of such processes, a critical examination and appreciation of the inherent difficulties of historical inference and challenges specific to testing phylogeographical hypotheses are essential. As the field of statistical phylogeography continues to take shape many difficulties have been resolved. Nonetheless, careful attention to the complexities of testing historical hypotheses and further theoretical developments are essential to improving the accuracy of our conclusions about a species’ history.

Does niche divergence accompany allopatric divergence in Aphelocoma jays as predicted under ecological speciation? Insights from tests with niche models.

The role of ecology in the origin of species has been the subject of long‐standing interest to evolutionary biologists. New sources of spatially explicit ecological data allow for large‐scale tests of whether speciation is associated with niche divergence or whether closely related species tend to be similar ecologically (niche conservatism). Because of the confounding effects of spatial autocorrelation of environmental variables, we generate null expectations for niche divergence for both an ecological‐niche modeling and a multivariate approach to address the question: do allopatrically distributed taxa occupy similar niches? In a classic system for the study of niche evolution—the Aphelocoma jays—we show that there is little evidence for niche divergence among Mexican Jay (A. ultramarina) lineages in the process of speciation, contrary to previous results. In contrast, Aphelocoma species that exist in partial sympatry in some regions show evidence for niche divergence. Our approach is widely applicable to the many cases of allopatric lineages in the beginning stages of speciation. These results do not support an ecological speciation model for Mexican Jay lineages because, in most cases, the allopatric environments they occupy are not significantly more divergent than expected under a null model.

TESTS OF PLEISTOCENE SPECIATION IN MONTANE GRASSHOPPERS (GENUS MELANOPLUS) FROM THE SKY ISLANDS OF WESTERN NORTH AMERICA

Abstract. There has a been a resurgence of debate on whether the Pleistocene glaciations inhibited speciation. This study tests a model of Pleistocene speciation, estimating the phylogenetic relationships and divergence times of 10 species of montane grasshoppers, genus Melanoplus, using 1300 bp of the mitochondrial gene cytochrome oxidase I (COI). Based on average pairwise distances (corrected for multiple substitutions using Kimuras two‐parameter model), all species appear to have originated within the Pleistocene. Sequence divergences between species are less than 4%, corresponding to divergence times less than 1.7 million years ago. Branching patterns among the species suggest that speciation was associated with more than one glacial‐interglacial cycle. A likelihood‐ratio test rejected a model of simultaneous species origins, the predicted branching pattern if species arose from the fragmentation of a widespread ancestor. These grasshoppers live in an area that was previously glaciated and, as inhabitants of the northern Rocky Mountain sky islands, underwent latitudinal and probably altitudinal shifts in distribution in response to climatic fluctuations. Given the repeated distributional shifts and range overlap of the taxa, there most likely has been ample opportunity for population mixing. However, despite periodic glacial cycles, with more than 10 major glaciations over the past million years and climatic fluctuations over as short a time scale as 103 to 104 years, the dynamic history of the Pleistocene did not preclude speciation. Although relationships among some taxa remain unresolved, these grasshopper species, even with their recent origins, exhibit genetic coherence and monophyletic or paraphyletic gene trees. The frequency of glacial cycles suggests that the speciation process must have been extremely rapid. These species of grasshoppers are morphologically very similar, differing primarily in the shape of the male genitalia. These characters are posited to be under sexual selection, may play an important role in reproductive isolation, and are known to diverge rapidly. This suggests the rapidity of evolution of reproductive isolation may determine whether species divergences occurred during the Pleistocene glaciations.

Calibrating divergence times on species trees versus gene trees: implications for speciation history of Aphelocoma jays

Estimates of the timing of divergence are central to testing the underlying causes of speciation. Relaxed molecular clocks and fossil calibration have improved these estimates; however, these advances are implemented in the context of gene trees, which can overestimate divergence times. Here we couple recent innovations for dating speciation events with the analytical power of species trees, where multilocus data are considered in a coalescent context. Divergence times are estimated in the bird genus Aphelocoma to test whether speciation in these jays coincided with mountain uplift or glacial cycles. Gene trees and species trees show general agreement that diversification began in the Miocene amid mountain uplift. However, dates from the multilocus species tree are more recent, occurring predominately in the Pleistocene, consistent with theory that divergence times can be significantly overestimated with gene‐tree based approaches that do not correct for genetic divergence that predates speciation. In addition to coalescent stochasticity, Haldanes rule could account for some differences in timing estimates between mitochondrial DNA and nuclear genes. By incorporating a fossil calibration applied to the species tree, in addition to the process of gene lineage coalescence, the present approach provides a more biologically realistic framework for dating speciation events, and hence for testing the links between diversification and specific biogeographic and geologic events.

Shifting distributions and speciation: species divergence during rapid climate change

Questions about how shifting distributions contribute to species diversification remain virtually without answer, even though rapid climate change during the Pleistocene clearly impacted genetic variation within many species. One factor that has prevented this question from being adequately addressed is the lack of precision associated with estimates of species divergence made from a single genetic locus and without incorporating processes that are biologically important as populations diverge. Analysis of DNA sequences from multiple variable loci in a coalescent framework that (i) corrects for gene divergence pre‐dating speciation, and (ii) derives divergence‐time estimates without making a priori assumptions about the processes underlying patterns of incomplete lineage sorting between species (i.e. allows for the possibility of gene flow during speciation), is critical to overcoming the inherent logistical and analytical difficulties of inferring the timing and mode of speciation during the dynamic Pleistocene. Estimates of species divergence that ignore these processes, use single locus data, or do both can dramatically overestimate species divergence. For example, using a coalescent approach with data from six loci, the divergence between two species of montane Melanoplus grasshoppers is estimated at between 200 000 and 300 000 years before present, far more recently than divergence estimates made using single‐locus data or without the incorporation of population‐level processes. Melanoplus grasshoppers radiated in the sky islands of the Rocky Mountains, and the analysis of divergence between these species suggests that the isolation of populations in multiple glacial refugia was an important factor in promoting speciation. Furthermore, the low estimates of gene flow between the species indicate that reproductive isolation must have evolved rapidly for the incipient species boundaries to be maintained through the subsequent glacial periods and shifts in species distributions.

Estimating species trees: Methods of phylogenetic analysis when there is incongruence across genes

Discord among the gene trees of multilocus data has motivated the development of phylogenetic approaches that account for gene-tree heterogeneity in the estimation procedure. Rather than equating a gene tree with the phylogenetic history, the new approaches explicitly consider the relationships between gene trees and the underlying history of species divergence, providing direct estimates of species trees (Fig. 1). The inherent appeal of these approaches is 2-fold. Incorporating information contained in the distribution of gene trees not only extracts phylogenetic signal, but modeling the relationship between the gene trees embedded in a species tree also reveals the biological processes that have influenced the diversification history and shaped organismal genomes. In contrast, ignoring the variance in genealogical histories (e.g., concatenating loci into a single supermatrix) disregards an inescapable biological reality—gene trees differ for a variety of reasons (reviewed in Maddison 1997; Degnan and Rosenberg 2009). As such, when the natural variation in gene trees is not taken into account during phylogenetic estimation, the reliability of inferences from such approaches is drawn into question (Degnan and Rosenberg 2006; Kubatko and Degnan 2007; Huang and Knowles 2009), historical scenarios with recently diverged taxa that have not reached reciprocal monophyly has become intractable (Carstens and Knowles 2007), and interpretation of the support for bipartitions across taxa is problematic (Mossel and Vigoda 2005).